Method for in vitro preconditioning of myoblasts before transplantation

ABSTRACT

Methods of pretreating healthy donor&#39;s myoblast cultures with growth or trophic factors on transplantation into subjects suffering from myopathic conditions such as muscular dystrophy. Compositions comprising myoblasts and fusion-promoting metalloproteases can be transplanted. Alternatively, myoblasts can be transplanted along with an agent inducing the expression of a fusion-promoting metalloprotease, or a composition comprising genetically-modified myoblasts capable of expressing a fusion-promoting metalloprotease can be transplanted.

This is a divisional of U.S. patent application Ser. No. 10/105,815,filed Mar. 21, 2002, which in turn was a continuation of U.S. Patentapplication Ser. No. 09/284,605, filed Jun. 9, 1999, which in turn was aU.S. national phase under 35 U.S.C. §371 of PCT/CA97/00774, which wasfiled on Oct. 17, 1997. Priority under 35 U.S.C. §120 is claimed to Ser.No. 10/105,815, to Ser. No. 09/284,605, and to PCT/CA97/00774. Priorityunder 35 U.S.C. §119(e) is claimed through PCT/CA97/00774 to U.S.provisional application Ser. No. 60/028,692, which was filed on Oct. 18,1996. The disclosure of each of the foregoing applications isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention is a method for preconditioning healthy donor'smyoblasts in vitro before transplantation thereof in compatible patientssuffering of recessive myopathies, particularly of muscular dystrophy.This in vitro preconditioning improves the success of thetransplantation while not requiring an in vivo preconditioning of thepatient's muscle by irradiation or by administering muscular toxin.

BACKGROUND OF THE INVENTION

Duchenne muscular dystrophy (DMD) is a progressive disease characterizedby the lack of dystrophin under the sarcolemmal membrane^(6,19,28,37) .One possible way to introduce dystrophin in the muscle fibers of thepatients to limit the degeneration is to transplant myoblasts obtainedfrom normal subjects^(30,34,35). Several groups have tried myoblasttransplantations to DMD patients but poor graft success wasobserved^(17,22,24,38). Even in experimental myoblast transplantationusing mdx mice, an animal model of DMD^(10,25,29), large amount ofdystrophin-positive fibers were observed only when nude mdx mice werepreviously irradiated to prevent regeneration of the muscle fibers byhost myoblasts^(32,43). High percentage of dystrophin-positive fiberswas also observed in mdx mice immunosuppressed with FK 506 and in SCIDmice, in both cases muscles were previously damaged by notexin injectionand irradiated^(23,27). These results indicate that to obtain successfulmyoblast transplantation, it is necessary to have not only animmunodeficient mouse or a mouse adequately immunosuppressed but also ahost muscle which has been adequately preconditioned. It is, however,impossible in clinical studies to use damaging treatments such asmarcaine, notexin and irradiation. If good myoblast transplantationresults can be obtained without using such techniques, this would bevery helpful for myoblast transplantation in humans.

Recently there has been an increasing interest on the effects of basicfibroblast growth factor (bFGF) and other growth factors on myoblastcultures and myoblast cell lines^(1,4,5). Basic FGF has been reported toboth stimulate proliferation and inhibit differentiation of skeletalmyoblasts in vitro^(5,6). Other growth or trophic factors like insulingrowth factor I, transferrin, platelet-derived growth factor, epidermalgrowth factor, adrenocorticotrophin and macrophage colony-stimulatingfactor as well as C kinase proteins activators or agonists by which theeffect of bFGF is mediated²⁰ may also have similar or even bettereffects than bFGF on the success of myoblast transplantation⁷. The useof these stimulating properties to enhance the success oftransplantation by in vitro preconditioning of donor's cells and toreplace at least partially the use of previously known methods of invivo preconditioning of recipients' cells has never been suggestedbefore.

Furthermore, it has been recently published by Overall and Sodek (1996)that concanavalin A increased the secretion of metalloproteases byfibroblasts. Since these enzymes are believed to be present in primarymyoblasts cultures, and since they may be responsible for thedegradation of the extracellular matrix, it would be desirable toprecondition the myoblasts in the presence of both a growth factor andan inducer of the production of metalloproteases, to increase thedistance of migration of the transplanted myoblasts and to increase thenumber of fused myoblasts expressing muscle functional proteins. Anattractive alternative would be to use donor myoblasts wherein a geneexpressing a metalloprotease is inserted.

Metalloproteases are enzymes necessary for tumor invasion, for cellmigration⁴⁵, and for restructuration of extracellular matrix duringnormal tissue remodelization⁴⁶. Matrilysine and gelatinase A aremetalloproteases involved in tissue invasion of a plurality of cancertypes⁴⁷. The presence of gelatinase A in its active form has beencorrelated with the generation of new muscle fibers, during muscledegeneration-regeneration process⁴⁸. It has been shown that the activityof gelatinase A can induce cell migration by cleaving laminin-5, anextracellular matrix component, thereby exposing a pro-migratory krypticsite

From the foregoing, it is really apparent that a compound capable ofstimulating the expression of a metalloproteases involved in anextra-cellular restructuration, such as phorbol ester or concanavalin A,would be useful to increase the success of transplantation of myoblasts.Since metalloproteases appear to be secreted in the culture medium, itwould also be useful to test if metalloproteases such as matrilysine,gelatinase A, or other metalloproteases of the same class, could beinjected directly with myoblasts in recipient muscle for the samepurpose.

SUMMARY OF THE INVENTION

The present invention relates to a method of in vitro preconditioning ofmyoblasts harvested from healthy donor's biopsy prior to theirtransplantation in patients affected by recessive myopathies,particularly by Duchenne muscular dystrophy (DMD). In a DMD animal model(mdx), compatible donor mouse myoblasts were grown in culture withmuscular growth or trophic factors, particularly, basic FibroblastGrowth Factor (bFGF), before transplanting them in muscles of mdx micewithout any previous damaging treatment. A four fold increase in thepercentage of muscle fibers expressing dystrophin, which is indicativeof functional muscle cells, was obtained with pretreatment with bFGF.These experimental results are expected to verify in naturally occurringdystrophy or other types of recessive myopathies in animal and humansubjects, since the mdx mouse is an animal model wherein musculardystrophy is naturally occurring.

Furthermore, culturing the myoblasts in the presence of concanavalin Aduring two to four days prior to transplantation increases by 3 to 4fold the distance of migration of the transplanted cells into therecipient tissue. Another inducer of the expression of metalloproteases,phorbol ester has been also used and reproduced the same result as forconcanavalin A (increase migration and increased number of fused cellsexpressing a reporter gene). Recombinant myoblasts expressingmetalloproteases also produced the same result.

It is therefore an object of the invention to provide a method whereincultured myoblasts are transplanted in the presence of ametalloprotease. The production of metalloproteases may be inducedduring the period of culturing of primary myoblast cultures with orwithout the preconditioning step in the presence of muscle growthfactor. Alternatively, the metalloproteases may be expressed byrecombinant myoblasts or injected concurrently with the transplantedmyoblasts. Transplantation of cells along with a matrix degrading amountof metalloproteases and transplantation of recombinant cells expressingthese enzymes are not limited to myoblasts, but could rather be adaptedto any type of transplantated cells.

In accordance with the present invention is provided a method ofincreasing the number of transplanting donor's myoblasts which arecapable of fusing with the myoblasts of a recipient individual sufferingof a myopathies, which comprises the steps of: growing said donor'smyoblasts in an appropriate culture medium in the presence offibroblasts and of an agent inducing an increased secretion of an enzymeinvolved in extracellular matrix destruction prior to injecting saidmedium, donor's myoblasts and fibroblasts to said recipient individual.

Alternatively is provided a method, wherein the donor's myoblasts arerecombinant myoblasts expressing a gene coding for said enzyme.

Is further provided a method which comprises reproducing one of theabove methods, and combining to the inducer agent a growth or trophicfactor to increase the multiplication of said healthy myoblasts.

In a specific embodiment, said myopathy is Duchenne muscular dystrophy.

In a preferred embodiment, donor's myoblasts consist of a primarymyoblast culture obtained from culturing of an enzymatic cell dispersionof donor's muscle biopsy.

It has been observed that growing of primary cultures of donor'smyoblasts, which contain fibroblasts, in the presence of a growth ortrophic factor is an in vitro preconditioning step that replaces atleast in part an in vivo preconditioning of said recipient individual'smuscular tissue by irradiation or by administering a muscular toxin.

The growth or trophic factor is selected from the group consisting ofbasic fibroblast growth factor (bFGF), insulin growth factor I,transferrin, platelet-derived growth factor, epidermal growth factor,adrenocorticotrophin, macrophage colony-stimulating factor, proteinkinase C activators, agonists thereof, and combinations thereof.

In a preferred embodiment, the growth or trophic factor is basicfibroblast growth factor (bFGF).

In a more preferred embodiment, the primary myoblast culture is grown inthe presence of 100 ng of recombinant human bFGF per milliliter ofculture medium for a period of time of about 48 hours beforetransplantation, whereby a four fold increase of the number offunctional muscular cells is obtained.

In still a preferred embodiment, the enzyme involved in theextracellular matrix destruction is a metalloprotease such asmatrilysine and gelatinase A and the inducer agent is phorbol ester orconcanavalin A.

In a most preferred embodiment, the inducing agent is concanavalin A(Con A)

In a specific embodiment, growing primary myoblast cultures in thepresence of 20 μg/ml of Con A for 48 hours resulted in a 3-4 foldincrease of the migration distance of transplanted myoblasts and offused myoblasts.

In the most preferred embodiment, primary myoblast cultures are culturedfor two days in the presence of both Con A and bFGF, which would resultin a superior transplanting success when compared to each treatmentalone.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is described hereinbelow with reference to thedrawings, the purpose of which is to illustrate this invention ratherthan to limit its scope.

FIG. 1 shows cross sections of TA muscle of mdx mice 28 days afterinjection of the transgenic myoblasts. Pairs of serial sections from 3different muscles of three mice are illustrated. Panels a and billustrate sections of muscles injected with myoblasts grown withoutbFGF. Panels c to f illustrate sections of muscles injected withmyoblasts grown with bFGF. In each pair, one section was stained forβ-galactosidase (panels a, c and e). The other section of the pair wasimmunostained for dystrophin (panels b, d and f). The muscles injectedwith myoblasts grown in presence of bFGF contained much moreβ-galactosidase and dystrophin positive fibers than muscles injectedwith myoblasts grown without bFGF. Most muscle fibers expressingβ-galactosidase were dystrophin-positive. In each pair of panels, thesame muscle fibers are identified by the same numbers. Scale bar is 100μm.

FIG. 2 shows the number of muscle fibers positive for X-Gal countedafter an injection of 500,000 donor's cells in one site of the tibialisanterior of recipient mice. Imm 7 neo: expresses neomycin. Imm 7Matrilysine: expresses neomycin and matrilysine. Tn I βgal: untreatedtransgenic mouse myoblasts expressing β-Gal. Tn I βGal+TPA: transgenicmouse myoblasts /β-Gal treated with phorbol ester.

DETAILED DESCRIPTION OF THE INVENTION

Although the present trend on research for the treatment of DMD seems tobe towards gene therapy, rather than cell therapy, there is still agreat deal of work to be done in animal models before either approach,or a mixture of both approaches will be required for the treatment ofinherited myopathies such as DMD^(32,34).

No satisfactory level of dystrophin expression was obtained followingmyoblast transplantation not only in clinical trials but also in animalexperiments not using irradiation³³ combined with marcaine or notexindestruction of the muscle^(26,27). These techniques are, however, toodamaging, too invasive or too risky to be used in clinical trials. BasicFGF has been reported to both stimulate proliferation and inhibitdifferentiation of skeletal myoblasts by suppressing muscle regulatoryfactors such as MyoD and myogenin^(12,41). Expression of bFGF has beenexamined in regenerating skeletal muscles by immunohistochemistry and insitu hybridization, and found to be up-regulated compared to non-injuredmuscles^(3,11). Increased skeletal muscle mitogens have also beenobserved in homogenates of regenerating muscles of mdx mice³. There areincreased levels of bFGF in extracellular matrix of mdx skeletalmuscles¹³, mdx satellite cells associated with repair³ and such cellsrespond more sensitively to exogenous addition of bFGF¹⁴. There is ahigh degree of homology between bFGF from various species² thereforerecombinant human bFGF is active on mouse cells⁹. In the present seriesof experiments, myoblasts were pretreated with recombinant human bFGF toincrease their proliferation and to verify whether such treatment whichis less invasive could have beneficial effects on myoblasttransplantation.

In our experiments, primary myoblast cultures from the same donors weregrown with or without bFGF and transplanted simultaneously to bothtibialis anterior (TA) muscles of the same mice. This seems to be a goodmodel to verify the effect of bFGF because the same primary myoblastcultures, the same grafting conditions and the same immunosuppressivestate were used. Comparing both TA muscles, in all treated mdx mice, thepercentage of β-galactosidase-positive fibers (this enzyme being areporter gene) were significantly higher in left TA muscles cultures(with bFGF) than in right TA muscles cultures (without bFGF). In themuscles grafted with myoblasts grown with bFGF, the average percentageof hybrid fibers was 34.4%, with two muscles containing over 40% ofdonor or hybrid fibers. These are the best results ever reportedfollowing myoblast transplantation without notexin or irradiationtreatment.

In the present study, myoblasts were incubated with bFGF during 48 hoursand about 5 millions of these cells (about 1.75 million myogenic cells)were injected in one TA muscle. The same number of myoblasts notincubated with bFGF was injected in the control contralateral TA muscle.The higher percentage of β-galactosidase/dystrophin-positive fibers wastherefore not the consequence of a higher proliferation of the myoblastsin vitro before the transplantations.

Our in vitro results indicate that an incubation during 2 days with bFGFdid not significantly modify the total number of cells and thepercentage of myogenic nuclei. Basic FGF did, however, significantlyinhibit the fusion of myoblasts in vitro. This resulted in a small butsignificant increase (35%) of the percentage of myoblasts amongmononuclear cells. This increase seems too small to account alone forthe more than four fold increase of effectiveness of myoblasttransplantation produced by bFGF. Recently both Partridge⁷ andKarpati's²⁴ group reported that a high percentage (up to 99% inPartridge's results) of the myoblasts injected in a mouse die within 5days. This dramatic result does not seem attributable to immunologicalproblems since it was observed following autotransplantation²⁴ ortransplantation in nude mice⁷. In our experiments, although slightlymore cells survived three days post-transplantation for the culturestreated with bFGF, the difference did not reach a significant level anddoes not seem to account alone for the 4 fold beneficial effect observed30 days post transplantation.

Basic FGF is thought to regulate myogenesis during muscle developmentand regeneration in vivo³. The increase percentage of muscle fiberscontaining the donor gene produced by the addition of bFGF may seemsurprising since bFGF was reported to inhibit differentiation ofmyoblasts in vitro^(1,13) Basic FGF is, however, one of many growthfactors which are liberated following muscle damage⁷. These factors, alltogether, certainly increase myoblast proliferation and eventuallymuscle repairs. We have also observed that following a two dayincubation with bFGF of primary myoblast cultures, myoblast fusionoccurred within a few days after removal of bFGF (data not shown). Theinhibition by bFGF on myoblast fusion is therefore not irreversible.Basic FGF is already at an increased level in mdx muscle, therefore itis not surprising that direct intramuscular injection did not increasethe fusion of the donor myoblasts with the host fibers. In fact, bFGFinjected directly in the muscle probably stimulates the proliferation ofthe host as well as the donor myoblasts and therefore do not favour thedonor myoblasts. On the contrary, preliminary stimulation by bFGF of thedonor myoblasts in culture may favour these myoblasts to proliferatemore and eventually participate more to muscle regeneration than thehost myoblasts. Though bFGF stimulates the fibroblasts, which aninconvenience for primary myoblast cultures, incubation of myoblastprimary culture during only 48 hours with bFGF did not adversely affectour transplantation results and did on the contrary improve them. Ifprimary myoblast cultures were made fibroblast-free by sub-cloning, itis envisageable to precondition the donors' myoblasts for a longer timeand increasing this way the number of cells to be transplanted from arelatively small biopsy.

Although the results obtained following transplantation of myoblastsgrown with bFGF are not as good than those obtained using irradiationand notexin²⁷, these results are nevertheless important because notechnique to destroy the muscles was used. The proposed in vitropreconditioning method might therefore be used in complete replacementof such in vivo damaging pretreatment of recipient cells, or at least inpartial replacement thereof, which will result in a substantialdiminution of undesirable effects. The effects of many growth factorsand trophic factors on myoblast culture have been reported, it ispossible that other factors such as insulin growth factor I,transferrin, platelet-derived growth factor, epidermal growth factor,adrenocorticotrophin and macrophage colony-stimulating factor may alsohave similar or even better effects than bFGF on the success of myoblasttransplantation⁷. Furthermore, since the effect of bFGF is mediated byproteins kinase C, pharmacological agents used to enhance the activityof these enzymes (like phorbol esters) or mimicking the effect thereof(agonists) might also be used for preconditioning myoblasts. Therefore,at least one of these factors can be used alone or in combination withor without bFGF to enhance the success of myoblast transplantation.While the mechanism involved remains speculative, bFGF seems to improvethe long term viability, multiplication and fusion of myoblasts. Ourresults suggest that pretreatment of myoblasts with bFGF may be oneprocedure that may increase the success of myoblast transplantation inDMD patients.

EXAMPLES

The present invention will be further described by way of the followingExamples, the purpose of which is to illustrate this invention ratherthan to limit its scope.

Example 1 Materials and Methods

Myoblast Cultures

Primary myoblast cultures were established from muscle biopsies ofnewborn transgenic mice²⁶. The founder mouse (TnI Lac Z1/29) wasprovided by Dr. Hasting (McGill University, Montreal, Canada) onto theCD1 background and was reproduced in our laboratory. This transgenicmouse expresses the β-galactosidase gene under the control of thepromoter of the quail fast skeletal muscle troponin I gene¹⁶. Bluemuscle fibers are revealed in these transgenic mice following incubationwith a substrate, 5-brom-4-chlor-3-indolyl-β-D-galactopyronoside (X-gal)(Boehringer Mannheim Canada, Laval, Canada). Before starting myoblastcultures, it was necessary to identify transgenic newborns by X-galstaining of a small muscle biopsy because heterozygote transgenic micewere used as parents. Myogenic cells were released from skeletal musclefragments of the transgenic newborns by serial enzyme treatments. First,a one hour digestion was done with 600 U/mi collagenase (Sigma,St-Louis, Mo., USA). This was followed by a 30 minute incubation inHanck's balanced salt solution (HBSS) containing 0.1% w/v trypsin (GibcoLab, Grand Island, N.Y., USA). Satellite cells were placed in 75 cm²culture flasks (Coster, Cambridge, Ma., USA) in proliferating medium,i.e. 199 medium (Gibco Lab.) with 15% fetal bovine serum (Gibco Lab.),1% penicillin (10,000 U/ml) and 1% streptomycin (10,000 U/ml).

Myoblast Transplantation

One day after starting culture, the culture medium of some flasks wasreplaced by medium containing 100 ng/ml human recombinant bFGF (Sigma).Three days after starting culture, myoblasts were detached from theflasks with 0.1% trypsin followed by three suspensions in HBSS andcentrifugations (6500 RPM, 5 minutes). The final cell pellet was dilutedin only 40 μl of HBSS.

Seventeen C57BL/10ScSn mdx/mdx mice (mdx mice) approximately one monthold were used for this experiment. This work was authorized andsupervised by the Laval University Animal Care Committee and wasconducted according to the guidelines set out by the Canadian Council ofAnimal Care.

The mdx mice were divided in three groups. Six mdx mice of one groupwere grafted in both tibialis anterior (TA) muscles: myoblasts grownwith bFGF were injected in the left TA and myoblasts grown without bFGFwere injected in the right TA. Myoblasts grown without bFGF wereinjected in only the left TA of six other mdx mice. These six mdx micewere then injected intramuscularly four times (after grafting 0, +1, +4and +6 days) either with 10 μl of bFGF (100 ng/ml, 3 mice) or with 10 μlof HBSS (3 mice). The last five mice were grafted in both TA muscle withnormal CD1 mouse myoblasts infected with replication defectiveretroviral vector LNPOZC7 (gift from Dr C. Cepko, Harvard, Boston, Ma.)which contains the LacZ gene. The left TA muscles were injected with 4million myoblasts grown with bFGF, while the right TA muscles wereinjected with 4 million myoblasts grown without bFGF. Three days aftergrafting, these 5 mice were sacrificed to detect the number ofβ-galactosidase positive cells which survived in each TA muscle. Thenumbers of β-galactosidase positive cells were counted in 8 μm sectionsobtained at every 160 μm throughout the muscle. The total number ofcells counted was multiplied by 20 to obtain an estimate of the numberof surviving cells and a correction was made to account for thepercentage of unlabelled cells in cultures with and without bFGF.

For the myoblast injection, the mice were anesthetized with 0.05 ml of asolution containing 10 mg/ml of ketamine and 10 mg/ml xylazine. The skinwas opened to expose the TA muscle. The myoblast suspension was taken upinto a glass micropipette with 50 μm tip (Drummond Scientific Company,Broomall, Pa., USA). The TA muscle was injected at 10 sites with a totalof about 5 million cells. The skin was then closed with fine sutures. FK506 (Fujisawa Pharmaceutical Co Ltd, Osaka, Japan) was administered at2.5 mg/kg to immunosuppress the animals. Alternatively, theimmunosuppressive treatment can be made by other pharmacological agentslike cyclosporin (Sandoz), RS61443 (Syntex) or rapamycin(Wyeth-Ayerst)⁴².

Muscle Examination

Three or twenty-eight days after myoblast transplantation, the mice weresacrificed by intracardiac perfusion with 0.9% saline under deepanesthesia of 10 mg/ml ketamine and 10 mg/ml xylazine. The TA muscleswere taken out and immersed in a 30% sucrose solution at 4

C for 12 hours. The specimens were embedded in OCT (Miles Inc, Elkhart,In. USA) and frozen in liquid nitrogen. Serial cryostat sections (8 μm)of the muscles were thawed on gelatin coated slides. These sections werefixed in 0.25% glutaraldehyde and stained in 0.4 mM X-gal in a dark boxovernight (12 hours) at room temperature to detect the muscle fiberscontaining β-galactosidase. Dystrophin was detected on adjacent cryostatsections by an immunoperoxidase technique with a sheep polyclonalantibody against the 60 KD dystrophin fragment (R27, Genica Co, Boston,Mass., USA) and the peroxidase activity was revealed by a 10 minuteincubation with 3,3′ diaminobenzidine (DAB, 0.5 mg/ml, Sigma) andhydrogen peroxidase (0.015%).

Desmin Staining

The primary cultures were washed with PBS and fixed with 100% methanolat −4

C. They were then washed again 3 times with PBS and incubated 1 hr witha mAb anti-human desmin (Dako, Copenhagen, Denmark) diluted 1/50 withPBS containing 1% blocking serum (i.e. 0.33% rabbit serum, 0.33% horseserum and 0.33 fetal calf serum). They were washed 3 times with PBS with1% blocking serum and incubated 1 hr with a 1/100 dilution (in PBS with1% blocking serum) of a rabbit anti-mouse immunoglobulin (Dako).Following 3 washes with PBS, the peroxidase activity was revealed withDAB as for dystrophin immunohistochemistry.

RESULTS

Myoblasts from muscle biopsies of transgenic mice expressingβ-galactosidase under a muscle specific promoter were grown with orwithout bFGF and injected in mdx muscles not previous irradiated ordamaged with notexin. A month later, the animals were sacrificed and theinjected muscles were examined for the presence of β-galactosidase anddystrophin. Many positive muscle fibers were observed. In our previousexperiments, muscles of mdx mice which did not receive injections oftransgenic myoblasts remained completely devoid ofβ-galactosidase-positive fibers²². Therefore allβ-galactosidase-positive muscle fibers observed in grafted mdx musclesare resulting from the fusion of some donor myoblasts among themselves(donor's fibers) or with the host myoblasts (hybrid fibers). In serialmuscle sections, most of the β-galactosidase-positive muscle fibers wereobserved to be also dystrophin-positive (FIG. 1). In all biopsied TAmuscles, the number of β-galactosidase-positive muscle fibers wascounted and expressed as a percentage of the total number of fibers in across section. The sections containing of the maximum percentage ofβ-galactosidase-positive muscle fibers were selected for each muscle. Inmdx mice grated in both TA muscles, the percentage ofβ-galactosidase-positive muscle fibers in the left TA muscle (graftedwith myoblasts grown with bFGF) was compared with that in the right TAmuscle (grafted with myoblasts grown without bFGF) of the same mouse(Table 1). Without notexin and irradiation, only a low percentage ofhybrid or donor muscle fibers were observed in the right TA muscle i.e.the mean number of β-galactosidase-positive fibers per muscle crosssection was 156.3 giving a mean percentage of β-galactosidase-positivefibers of 8.396. The left TA muscles contained, however, significantlymore hybrid or donor muscle fibers, i.e. the mean number ofβ-galactosidase-positive fibers per muscle cross section was 773.7 thusgiving a mean percentage of β-galactosidase-positive fibers equal to34.4% (FIG. 1). This is more than a four fold increase in the efficacyof myoblast transplantation produced by the addition of bFGF to theculture medium.

We have also investigated whether the beneficial effect of bFGF could beobtained by injecting it directly in the muscle at 4 intervals aftermyoblast transplantation. No significant difference in the percentage ofhybrid or donor muscle fibers (i.e. β-galactosidase positive fibers) wasobserved between the groups which received intramuscular injections ofbFGF and those which received HBSS injections (control) (Table 2). Thepercentage of β-galactosidase positive muscle fibers was higherfollowing repeated injection of HBSS (14.8%) or of bFGF (15.9%) thanfollowing injection of myoblasts alone grown without bFGF (Table 1,8.3%). This may be due to damage produced by the repeated injectionswhich may increase the regeneration process.

It has been reported recently by Huard et al.²¹ and by Beauchamp etal.⁷, that a high percentage of the myoblasts injected in a muscle diedwithin the first few days following their transplantation. To examinewhether the increase efficiency of myoblast transplantation followingculture with bFGF could be due to a reduced cell death, we have labellednormal CD1 primary cultures grown with or without bFGF with a retroviralvector containing the β-galactosidase gene under an LTR promoter. Normalmyoblasts were labelled with a retroviral expressing β-galactosidasebecause only mature myoblasts and myotubes of transgenic TnI LacZ 1/29can express β-galactosidase. With labelling using a retroviral vector ahigher percentage of the cells in the primary culture expressed thereporter gene. The retrovirally labelled cells were then injected in amuscle of 5 mice. We examined the number of β-galactosidase positivecells 3 days after their transplantation. In all 5 mice, the number ofthe cells was not significantly higher in left TA muscles (with bFGF)(3.29±1.54×10⁵ cells) than in right TA muscles (without bFGF2.13±0.40×10⁵ cells). Note that since 4×10⁶ cells were injected in eachmuscle, there is only 5.3% of the injected cells surviving at 3 dayswithout bFGF while only 8.2% of the injected cells survived with bFGF.

To try to understand the beneficial effects of bFGF on myoblasttransplantation, we examined the effect of a short stimulation (2 days)with 100 ng/ml bFGF on primary myoblast cultures. The total number ofcells in each flask was not significant different (31.9±6.8×10⁶ withbFGF n=5, 30.0±5.8×10⁶ without FGF n=9, unpaired t-test: p=0.573). Themyoblasts and myotubes were then identified by revealing desmin byimmunoperoxidase. In these cultures, there was no difference in thepercentage of myogenic nuclei (nuclei in myoblasts and in myotubes)between the two groups of cultures (Table 3, line 1). More myogeniccells were however fused in the absence of bFGF (Table 3, line 2). Therewas an higher percentage of the total nuclei (including myoblasts,myotubes and fibroblasts) which were myoblast nuclei in culturescontaining bFGF (Table 3, line 3). The increase of myoblasts was moreclear when the percentage of myoblasts was calculated among mononuclearcells (excluding the myotubes) (Table 3, lines 4 and 5). This washowever only a 35% increase. TABLE 1 Effect of culture with or withoutbFGF on the formation of muscle fibers containing donor's gene in mdxmice no bFGF (right TA muscle) with bFGF (left TA muscle) No of No (%)of β-gal. No (%) of β-gal. mdx mice positive fibers positive fibers 1170(11.0) 514(19.3) 2 259(11.9) 438(20.4) 3 259(13.1) 1007(37.4)  457(4.1) 695(34.0) 5 139(6.1)  848(43.8) 6 54(3.6) 1140(51.7) Mean ± SD156.3 ± 91.5(8.3 ± 4.2)# 773.7 ± 275.8(34.4 ± 12.8)##Paired t-test indicated a significant difference (p < 0.05)

TABLE 2 Effects of bFGF on primary myoblast culture no bFGF with bFGF(mean ± SD) (mean ± SD) sign 1) % of myoblast and myotube 34.5 ± 5.335.1 ± 4.8 0.81 nucleic relative to total nuclei 2) % of myotube nucleirelative 40.8 ± 8.0 11.5 ± 6.6 0.0001 to total myotube and myoblastnuclei 3) % myoblast nuclei relative to 21.1 ± 3.6 30.9 ± 3.8 0.0001total nuclei 4) % myoblast nuclei relative to 23.9 ± 5.4 32.2 ± 4.10.001 non myotube nuclei 5) % of non-myoblast nuclei 76.1 ± 5.4 67.8 ±4.1 0.001 relative to non myotube nuclei

TABLE 3 Effect of intramuscular injections of bFGF in mdx mice No (5%)of β-gal. positive fibers Mean ± SD HBSS IM injections 1 180(12.4) 372.0± 172.8 (14.8 ± 2.9) 2 421(14.1) 3 515(18.0) bFGF IM injections 1176(7.4) 289.7 ± 167.5 (15.9 ± 8.4) 2 482(24.1) T test indicated nosignificant 3 211(16.3) difference (p > .05)

Example 2

The above results can be extrapolated to an in vivo utility and verifiedin patients suffering of muscular dystrophy. The healthy donors and DMDrecipients should be matched, if possible, upon their compatibility forthe MHC (HLA)-class I (A,B,C) and -class II (Dr) antigens. Thedystrophic patients should undertake an immunosuppressive treatment bybeing administered, for example, FK 506, cyclosporin, RS61443 orrapamycin. Donors' biopsy would then be treated substantially inaccordance with the procedures given in Example 1 with regard to micemyoblasts. The success of the transplantation might be monitored bymeasuring the incidentce of dystrophin-positive fibers from a biopsyobtained from the site of transplantation and by evaluating theresulting increase of muscular strength³⁹.

Example 3

Myoblasts infected with a retrovirus expressing a beta-galactosidasegene have been cultured for four days in the presence or absence of20μg/ml concanavalin A, a lectine which stimulates the expression ofmetalloproteases. These myoblasts were then injected in one single siteof the anterior tibialis muscle of eight mice, in order to verify thedegree with which the transplanted cells are capable of migratingthrough the recipient tissue. After thirty days, mice were sacrificedand the muscle tissue was harvested, frozen and 10 μm thick slices weremounted on slides. The presence of beta-galactosidase was revealed withX-Gal. Labelled cells were observed at a distance which is 3 to 4 foldgreater in the mice muscle treated with concanavalin A. Concanavalin Ais known to induce the secretion of metalloproteases by the fibroblastspresent in the primary cultures. Therefore, the presence ofmetalloproteases in the preconditioning medium or during thetransplantation is beneficial to the spreading of the transplanted cellsfrom the site of the injection through the recipient muscle tissue.

Three experiments confirmed that an increased expression of enzymesinvolved in the destruction of the extracellular matrix indeed increasesthe migration of myoblasts.

First Experiment

Myoblasts obtained from CMVLacZ mice (expressing a Beta-galactosidasegene) were cultured for two (2) days in the presence of twenty (20)μg/ml concanavalin A, a lectine which stimulates the expression ofmetalloproteases. When these myoblasts were injected in the anteriortibialis muscle of recipient mice, the migration of transplanted cellswas increased by three (3) fold comparatively to myoblasts having notreceived the concanavalin treatment.

Second Experiment

primary cultures of myoblasts were obtained from transgenic mice(TnI-βGal+TPA). Myoblasts were cultured for two (2) days in the presenceof phorbol ester (TPA 100 ng/ml) which also stimulates the expressionmetalloproteases. These treated myoblasts generated four times morefibers on an area four fold greater than for transplanted myoblastsinjected from a non-treated preparation (See FIG. 2; n=5 mice).

Third Experiment

To verify if metalloproteases are really involved in the increasedspread of transplanted myoblasts, we have stably transfected animmortalized myoblast cell line already expressing a β-Gal gene with anexpression vector comprising human matrilysine gene and a neomycineresistance gene⁵⁰. This transfection greatly increased the fusioncapacity in vitro and the generation of fibers in vivo. Expression ofβ-Gal gene was measured. The clone Imm 7 Matrilysine was compared to thesame clone (Imm7) transfected solely with the neomycine resistance gene.As seen from FIG. 2, the injection of 500,000 cells in the anteriortibialis muscle of recipient mice (non irradiated and non injected witha myotoxic agent such as notexin) resulted in the average presence (n=8mice) of 39 positive fibers for beta-galactosidase following theinjection of the clone expressing matrilysine, while no fiber has beenobserved with the control clone, three (3) weeks after the injection.

Fourth Experiment

Tumoral myoblasts obtained from G8 mice transfected with the sameconstruct (matrilysine and neomycine recombinant) and labelled with afluorescent die (PKH26) have been allowed to migrate for eight (8) daysfollowing injection in the anterior tibialis muscle of recipient mice.The distance of migration was equivalent to three to four times thedistance observed with the control construct.

When myoblasts are cloned, which results in the removal of fibroblasts,it is believed that recombinant myoblasts expressing a metalloproteasegene product could be useful in increasing further the success oftransplantation per se or along with the increase due to thepreconditioning step with a growth factor.

Alternatively, metalloproteases could be directly injected with thedonor's myoblasts to increase the migration of the transplanted cells.

Although the present invention has been described hereinabove by way ofpreferred embodiments thereof, it can be modified, without departingfrom the spirit and nature of the subject invention as defined in theappended claims.

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1. A method for increasing the number of transplanted myoblasts whichmigrate into a recipient muscle tissue and which fuse either with themyoblasts or muscle fibers of said recipient muscle tissue, said methodcomprising transplanting into a patient affected by a recessive myopathya composition comprising myoblasts transformed with a gene constructcapable of expressing a metalloprotease involved in extracellular matrixdestruction.
 2. The method of claim 1, wherein the metalloprotease isGelatinase A, Matrilysine, or both.
 3. The method of claim 1, whereinthe expression of said metalloprotease is inducible.
 4. The method ofclaim 1, wherein said myoblasts to be transplanted are grown in vitro ina culture medium, suitable for growth of myoblasts, which comprises agrowth or trophic factor.
 5. The method of claim 4, wherein said growthor trophic factor is basic fibroblast growth factor.
 6. The method ofclaim 1, wherein the recessive myopathy affecting said patient isDuchenne muscular dystrophy.